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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CSDP (Construction of Self-Dividing Protocells)

Teaser

Summary

This project aimed to construct suitable compartments for synthetic cells. Such compartments should be biocompatible and be able to support a range of complex (bio)chemical processes. One of the most promising compartments are liposomes. Liposomes very much resemble the cell membrane and are composed of the same lipids. However, the formation of liposomes is typically uncontrolled and yields a highly polydisperse mixture. Importantly, the content of liposomes cannot be chosen, as it will be the same as the surrounding fluid. This complicates the study of reactions inside liposomes, as it is very difficult to guarantee that no reaction takes place in the exterior. The objective of this study was to find new methods for liposome formation. Using a microfluidic device, we were able to produce double emulsions (water in oil in water, W/O/W) which are precursors to liposomes. They key step is that the lipids stabilizing the oil/water interfaces must come together to form a lipid bilayer. To this end, the oil has to be removed. Typically, this will result in a large excess of lipid drying into a thin shell. However, by carefully controlled the kinetics of the evaporation of oil, and the concomitant changes in surface tension, we were able to dewet perfect liposomes from oil droplets. After establishing this technology, and expanding it to form liposomes-in-liposomes, hierarchical liposomes and vesosomes (i.e. all kind of architectures where different liposomes are placed in close proximity) we set about using these liposomes as containers for biochemical reactions. We established that it is possible to carry out enzymatic reactions as well as gene expression inside liposomes. By perforating the lipid bilayers using pore-forming proteins, we were able to extend reactions by feeding additional reagents from the outside. And finally, we were able to shrink and swell liposomes, thereby inducing phase transitions inside.

This project has contributed to efforts to build a synthetic cell. This is an effort that touches fundamental questions about life: what is life, and where does it come from. To answer these questions we must understand how life is organized, how reactions in tiny volumes are balanced and how we create functional systems out of relatively simple building blocks.

Work performed

Work performed includes a significant effort in the construction of so-called capilaary-in-capillary microfluidic devices for the production of double emulsions. Furthermore, a very detailed analysis of the different surface energies and the development of these surface energies during evaporation was made. This complete understanding of the physical chemistry of the system was used to produce perfect monodisperse liposomes. We then filled these liposomes with different enzymatic reactions as well as systems for cell free gene expression. In all case we were able to follow the progress of reactions inside liposomes using fluorescence microscopy.

Final results

The formation of perfect liposomes by controlling the dewetting of double emulsions formed using microfluidic devices is a major breakthrough. This method is now by far the best method to produce well-defined liposomes, and already other groups are copying, or intending to copy, this method. It will make future work on synthetic cells much easier, as it provides an experimental platform that is robust, scalable and compatible with a range of reactions one could wish to study.
This project has contributed to efforts to build a synthetic cell. This is an effort that touches fundamental questions about life: what is life, and where does it come from. To answer these questions we must understand how life is organized, how reactions in tiny volumes are balanced and how we create functional systems out of relatively simple building blocks. There is a growing consensus that Europe can play a leading role in addressing these age-old questions. Although the impact is initially of a scientific nature, the development of such disruptive ideas will no doubt lead to changes in the way we think about life, but also about how we \' fix\' life when it doesn\'t function as expected and this latter part will impact on the way we design drugs and materials to interact with living cells.